Delta 6 desaturases from primulaceae, expressing plants and pufa-containing oils

ABSTRACT

The present invention relates to an improved method for the specific production of unsaturated ω-3 fatty acids and a method for the production of triglycerides having an increased content of unsaturated fatty acids, in particular ω-3 fatty acids having more than three double bonds. The invention relates to the production of a transgenic organism, preferably a transgenic plant or a transgenic microorganism, having an increased content of fatty acids, oils or lipids having Δ6 double bonds due to the expression of a Δ6-desaturase from Primulaceae. The invention additionally relates to expression cassettes containing a nucleic acid sequence, a vector and organisms containing at least one nucleic acid sequence or an expression cassette. The invention further relates to unsaturated fatty acids and triglycerides having an increased content of unsaturated fatty acids and use thereof.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 10/504,424 filed Aug. 13, 2004, which is a national stageapplication (under 35 U.S.C. §371) of PCT/EP2003/01161 filed Feb. 6,2003, which claims benefit of United Kingdom application 0204676.1 filedFeb. 27, 2002. The entire content of each above-mentioned application ishereby incorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_List_(—)12810_(—)00851. The size of thetext file is 23 KB, and the text file was created on May 5, 2009.

FIELD OF THE INVENTION

The present invention relates to an improved method for the specificproduction of unsaturated ω-3 fatty acids and a method for theproduction of triglycerides having an increased content of unsaturatedfatty acids, in particular ω-3 fatty acids having more than three doublebonds. The invention relates to the production of a transgenic organism,preferably a transgenic plant or a transgenic microorganism, having anincreased content of fatty acids, oils or lipids having Δ6 double bondsdue to the expression of a Δ6-desaturase from Primulaceae.

The invention additionally relates to expression cassettes containing anucleic acid sequence, a vector and organisms containing at least onenucleic acid sequence or an expression cassette. The invention furtherrelates to unsaturated fatty acids and triglycerides having an increasedcontent of unsaturated fatty acids and use thereof.

BACKGROUND OF THE INVENTION

Fatty acids and triglycerides have numerous applications in the foodindustry, animal nutrition, cosmetics and in the drug sector. Dependingon whether they are free saturated or unsaturated fatty acids or aretriglycerides having an increased content of saturated or unsaturatedfatty acids, they are suitable for the most varied applications. Thus,for example, polyunsaturated fatty acids are added to infant formula toincrease its nutritional value. The various fatty acids andtriglycerides are mainly obtained from microorganisms such asMortierella or from oil-producing plants such as soybean, oilseed rape,sunflower and others, and generally occur in the form of theirtriacylglycerides. However, they may also be obtained from animals, e.g.fish. The free fatty acids are advantageously produced bysaponification.

Depending on application purpose oils containing saturated orunsaturated fatty acids are preferred, thus in human nutrition forexample, lipids containing unsaturated fatty acids, especiallypolyunsaturated fatty acids, are preferred since they have a positiveeffect on the level of cholesterol in the blood and hence on thepossibility of heart disease. They are employed in various dietary foodsor medicinal drugs.

On account of their positive properties there has been no shortage ofattempts in the past to make available genes which participate in thesynthesis of fatty acids or triglycerides for the production of oils invarious organisms having a modified content of unsaturated fatty acids.Thus, in WO 91/13972 and its US equivalent a Δ9-desaturase is described.In WO 93/11245 a Δ15-desaturase and in WO 94/11516 a Δ12-desaturase isclaimed. Other desaturases are described, for example, in EP-A-0 550162, WO 94/18337, WO 97/30582, WO 97/21340, WO 95/18222, EP-A-0 794 250,Stukey et al., J. Biol. Chem., 265, 1990: 20144-20149, Wada et al.,Nature 347, 1990: 200-203 or Huang et al., Lipids 34, 1999: 649-659. Todate, however, the various desaturases have been only inadequatelycharacterized biochemically since the enzymes in the form ofmembrane-bound proteins are isolable and characterizable only with verygreat difficulty (McKeon et al., Methods in Enzymol. 71, 1981:12141-12147, Wang et al., Plant Physiol. Biochem., 26, 1988: 777-792).Generally, membrane-bound desaturases are characterized by introductioninto a suitable organism which is then investigated for enzyme activityby means of analysis of starting materials and products. Δ6-Desaturasesare described in WO 93/06712, U.S. Pat. No. 5,614,393, U.S. Pat. No.5,614,393, WO 96/21022, WO 0021557 and WO 99/27111 and their applicationto production in transgenic organisms is also described, e.g. in WO9846763, WO 9846764 and WO 9846765. At the same time the expression ofvarious desaturases, as in WO 9964616 or WO 9846776, and the formationof polyunsaturated fatty acids is also described and claimed. Withregard to the effectiveness of the expression of desaturases and theireffect on the formation of polyunsaturated fatty acids it may be notedthat through expression of a single desaturase as described to date onlylow contents of Δ6 unsaturated fatty acids/lipids, such as by way ofexample gamma-linolenic acid and stearidonic acid, have been achieved.Furthermore, a mixture of ω-3 and ω-6 fatty acids was usually obtainedsince all Δ6-desaturases described so far converted, for example,linoleic acid (ω-6 fatty acid) as well as α-linolenic acid (ω-3 fattyacid).

Accordingly, there is still a great demand for new and more suitablegenes which encode enzymes which participate in the biosynthesis ofunsaturated fatty acids and make it possible to produce certain fattyacids specifically on an industrial scale without unwanted byproductsforming. In the selection of genes for biosynthesis two characteristicsabove all are particularly important. On the one hand, there is as evera need for improved processes for obtaining the highest possiblecontents of polyunsaturated fatty acids. On the other hand, the enzymesemployed should be highly specific to a certain substrate since as faras possible unwanted byproducts must not be produced which might havenegative or so far undiscovered physiological effects in humans oranimals due to food/feed intake.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to introducefurther genes of desaturase enzymes for the synthesis of polyunsaturatedfatty acids into the seeds of an oil seed and in doing so to prevent theproduction of unwanted byproducts. We have found that this object isachieved by the isolated nucleic acid sequences according to theinvention which encode polypeptides having Δ6-desaturase activity,wherein the Δ6-desaturases encoded by the nucleic acid sequencesspecifically convert ω-3 fatty acids. This object was achieved inparticular by cloning the isolated nucleic acid sequences according tothe invention, wherein the nucleic acid sequences encode a polypeptidehaving Δ6-desaturase activity, wherein the Δ6-desaturases encoded by thenucleic acid sequences specifically convert ω-3 fatty acids selectedfrom the group:

-   -   a) a nucleic acid sequence having the sequence depicted in SEQ        ID NO: 1 or SEQ ID NO: 3,    -   b) nucleic acid sequences which may be derived as a result of        the degenerated genetic code from the encoding sequence        contained in SEQ ID NO: 1 or SEQ ID NO: 3,    -   c) derivatives of the nucleic acid sequence depicted in SEQ ID        NO: 1 or SEQ ID NO: 3 which encode polypeptides using the amino        acid sequences depicted in SEQ ID NO: 2 or SEQ ID NO: 4 and have        at least 75% homology on the amino acid level with SEQ ID NO: 2        or SEQ ID NO: 4 and possess A 6-desaturase activity.

DETAILED DESCRIPTION OF THE INVENTION

The nucleic acid sequences according to the invention which encodepolypeptides having a Δ6-desaturase activity and originate from plants,advantageously Primulaceae such as Muscariodides or Aleuritia arespecific for the conversion of ω-3 fatty acids and thus they preferablyconvert by way of example α-linolenic acid and not linoleic acid whenthey are expressed in a heterologous system and both fatty acids areavailable in the organism. By this means e.g. stearidonic acid,eicosapentaenoic acid or docosahexaenoic acid are produced in the hostorganisms such as plants or microorganisms without formation ofarachidonic acid. This results in an advantageous synthesis of fattyacids of the ω-3 fatty acid family, while ω-6 fatty acids are scarcelyformed if they occur at all. The Δ6-desaturases according to theinvention exhibit a higher activity towards ω-3 fatty acids as comparedto ω-6 fatty acids by at least the factor 1.5, advantageously by at theleast the factor 2, preferably by at least the factor 3, particularlypreferably by at least the factor 4 and most particularly preferably byat least the factor 5. Due to this specificity the formation of unwantedfatty acids can be suppressed or completely prevented.

By derivative(s) of the sequences according to the invention is meant,for example, functional homologues of the polypeptides or enzymesencoded by SEQ ID NO: 1 or SEQ ID NO: 3 which exhibit the same saidspecific enzymatic activity. This specific enzymatic activity allowsadvantageously the synthesis of unsaturated fatty acids having more thanthree double bonds in the fatty acid molecule. By unsaturated fattyacids is meant in what follows diunsaturated or polyunsaturated fattyacids which possess double bonds. The double bonds may be conjugated ornonconjugated. The said sequences encode enzymes which exhibitΔ6-desaturase activity.

The enzyme according to the invention, Δ6-desaturase, advantageouslyintroduces a cis double bond into fatty acid residues of glycerolipidsat position C₆-C₇ (see SEQ ID NO: 1 and SEQ ID NO: 3). The enzymesadditionally have a Δ6-desaturase activity which advantageouslyintroduces exclusively a cis double bond into fatty acid residues ofglycerolipids at position C₆-C₇. This activity is also possessed by theenzymes having the sequences specified in SEQ ID NO: 1 and NO: 3 whichare monofunctional Δ6-desaturases.

The nucleic acid sequence(s) according to the invention (for purposes ofthe application the singular encompasses the plural and vice versa) orfragments thereof may advantageously bc used for isolating other genomicsequences via homology screening.

The said derivatives may be isolated, for example, from other organisms,eukaryotic organisms such as plants, especially mosses, dinoflagellatesor fungi.

Allele variants include in particular functional variants obtainable bydeletion, insertion or substitution of nucleotides in the sequencesdepicted in SEQ ID NO: 1 or SEQ ID NO: 3, the enzymatic activity of thederived synthesized proteins being retained.

Starting from the DNA sequence described in SEQ ID NO: 1 and SEQ ID NO:3 or parts of said sequences such DNA sequences can be isolated using,for example, normal hybridization methods or the PCR technique fromother eukaryotes such as those identified above for example. These DNAsequences hybridize under standard conditions with the said sequences.For hybridization use is advantageously made of short oligonucleotidesof the conserved regions, for example, which can be determined bycomparisons with other desaturase genes in the manner known to thoseskilled in the art. The histidine box sequences are advantageouslyemployed. However, longer fragments of the nucleic acids according tothe invention or the complete sequences may also be used forhybridization. Depending on the nucleic acid employed: oligonucleotide,longer fragment or complete sequence, or depending on which type ofnucleic acid, DNA or RNA, is used for hybridization these standardconditions vary. Thus, for example, the melting temperatures of DNA:DNAhybrids are approximately 10° C. lower than those of DNA:RNA hybrids ofthe same length.

By standard conditions is meant, for example, depending on the nucleicacid in question temperatures between 42° C. and 58° C. in an aqueousbuffer solution having a concentration of between 0.1 and 5×SSC(1×SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2) or additionally in thepresence of 50% formamide, such as by way of example 42° C. in 5×SSC,50% formamide. Hybridization conditions for DNA:DNA hybrids areadvantageously 0.1×SSC and temperatures between approximately 20° C. and45° C., preferably between approximately 30° C. and 45° C. For DNA:RNAhybrids the hybridization conditions are advantageously 0.1×SSC andtemperatures between approximately 30° C. and 55° C., preferably betweenapproximately 45° C. and 55° C. These specified temperatures forhybridization are melting temperature values calculated by way ofexample for a nucleic acid having a length of approximately 100nucleotides and a G+C content of 50% in the absence of formamide. Theexperimental conditions for DNA hybridization are described in relevantgenetics textbooks such as by way of example Sambrook et al., “MolecularCloning”, Cold Spring Harbor Laboratory, 1989, and may be calculated byformulae known to those skilled in the art, for example as a function ofthe length of the nucleic acids, the nature of the hybrids or the G+Ccontent. Those skilled in the art may draw on the following textbooksfor further information on hybridization: Ausubel et al. (eds), 1985,Current Protocols in Molecular Biology, John Wiley & Sons. New York,Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A PracticalApproach, IRL Press at Oxford University Press, Oxford; Brown (ed),1991, Essential Molecular Biology: A Practical Approach, IRL Press atOxford University Press, Oxford.

Furthermore, by derivatives is meant homologues of the sequences SEQ IDNO: 1 and NO: 3, for example eukaryotic homologues, truncated sequences,single-stranded DNA of the encoding and nonencoding DNA sequence or RNAof the encoding and nonencoding DNA sequence.

In addition, by homologues of the sequences SEQ ID NO: 1 and SEQ ID NO:3 is meant derivatives such as by way of example promoter variants.These variants may be modified by one or more nucleotide exchanges, byinsertion(s) and/or deletion(s) without, however, adversely affectingthe functionality or efficiency of the promoters. Furthermore, thepromoters can have their efficiency increased by altering their sequenceor be completely replaced by more effective promoters even of foreignorganisms.

By derivatives is also advantageously meant variants whose nucleotidesequence has been altered in the region from −1 to −2000 ahead of thestart codon in such a way that the gene expression and/or the proteinexpression is modified, preferably increased. Furthermore, byderivatives is also meant variants which have been modified at the 3′end.

By derivatives is also meant the antisense DNAs which may be employedfor inhibiting protein biosynthesis of the proteins according to theinvention. These antisense DNAs are numbered among the nonfunctionalderivatives according to the invention such as derivatives which exhibitno enzymatic activity. Other methods known to those skilled in the artfor the production of nonfunctional derivatives are what is known ascosuppression, the use of ribozymes and introns and the RNAi method.Ribozymes are catalytic RNA molecules having ribonuclease activity whichcan chop single-stranded nucleic acids, such as mRNA, with which theyare complementary. In this way, using these ribozymes (Haselhoff andGerlach, Nature, 334, 1988: 585-591) mRNA transcripts can becatalytically cleaved and, thus, the translation of this mRNA issuppressed. Ribozymes of this type can be specially tailored to theirpurpose (U.S. Pat. No. 4,987,071; U.S. Pat. No. 5,116,742 and Bartel etal., Science 261, 1993: 1411-1418). By this means, with the aid of theantisense DNA, fatty acids, lipids or oils having an increased contentof saturated fatty acids can be produced.

The nucleic acid sequence according to the invention which encodes aΔ6-desaturase may be produced by synthesis or obtained naturally orcontain a mixture of synthetic and natural DNA components as well asconsist of various heterologous Δ6-desaturase gene segments fromdifferent organisms. In general, synthetic nucleotide sequences areproduced with codons which are preferred by the corresponding hostorganisms, plants for example. This usually results in optimumexpression of the heterologous gene. These codons preferred by plantsmay be determined from codons having the highest protein frequency whichare expressed in most of the plant species of interest. An exampleconcerning Corynebacterium glutamicum is provided in Wada et al. (1992)Nucleic Acids Res. 20:2111-2118). Such experiments can be carried outusing standard methods and are known to the person skilled in the art.

Functionally equivalent sequences which encode the Δ6-desaturase geneare those derivatives of the sequence according to the invention whichdespite differing nucleotide sequence still possess the desiredfunctions, that is to say the enzymatic activity and specificselectivity of the proteins. Thus, functional equivalents includenaturally occurring variants of the sequences described herein as wellas artificial ones, e.g. artificial nucleotide sequences adapted to thecodon use of a plant which have been obtained by chemical synthesis.

In addition, artificial DNA sequences are suitable, provided, asdescribed above, they mediate the desired property, for example anincrease in the content of Δ6 double bonds in fatty acids, oils orlipids in organisms such as in a plant by overexpression of theΔ6-desaturase gene in crop plants. Such artificial DNA sequences canexhibit Δ6-desaturase activity, for example by back-translation ofproteins constructed by means of molecular modeling, or be determined byin vitro selection. Possible techniques for in vitro evolution of DNA tomodify or improve the DNA sequences are described in Patten, P. A. etal., Current Opinion in Biotechnology 8, 724-733 (1997) or in Moore, J.C. et al., Journal of Molecular Biology 272, 336-347 (1997).Particularly suitable are encoding DNA sequences which are obtained byback-translation of a polypeptide sequence in accordance with the codonuse specific to the host plant. Those skilled in the art familiar withthe methods of plant genetics can easily determine the specific codonuse by computer analyses of other known genes of the plant to betransformed.

Other suitable equivalent nucleic acid sequences which may be mentionedare sequences that encode fusion proteins, a component of the fusionprotein being a Δ6-desaturase polypeptide or a functionally equivalentpart thereof. The second part of the fusion protein can be, for example,another polypeptide having enzymatic activity or an antigenicpolypeptide sequence by means of which it is possible to demonstrateΔ6-desaturase expression (e.g. myc tag or his tag). Preferably, however,this is a regulatory protein sequence, such as by way of example asignal sequence for the endoplasmic reticulum (=ER) which directs theΔ6-desaturase protein to the desired point of action, or regulatorysequences which influence the expression of the nucleic acid sequenceaccording to the invention, such as promoters or terminators.

Advantageously, the Δ6-desaturase genes in the method according to theinvention may be combined with other genes for fatty acid biosynthesis.Examples of such genes are the acetyl transferases, other desaturases orelongases such as Δ4-, Δ5-, Δ6- or Δ8-desaturases or ω-3- and/orω-6-specific desaturases such as Δ12 (for C₁₈ fatty acids), Δ15 (for C₁₈fatty acids) or Δ19 (for C₂₂ fatty acids) or such as Δ5- orΔ6-elongases. For in vivo and especially in vitro synthesis combinationwith e.g. NADH cytochrome B5 reductases which can take up or releasereduction equivalents is advantageous.

By the amino acid sequences according to the invention is meant proteinswhich contain an amino acid sequence depicted in the sequences SEQ IDNO: 2 and SEQ ID NO: 4 or a sequence obtainable therefrom bysubstitution, inversion, insertion or deletion of one or more amino acidgroups, the enzymatic activities of the proteins depicted in SEQ ID NO:2 and NO: 4 being retained or not substantially reduced, that is theystill possess the same enzymatic activity. By “not substantiallyreduced” or “the same enzymatic activity” is meant all enzymes whichstill exhibit at least 10%, preferably 20%, particularly preferably 30%,of the enzymatic activity of the initial enzyme obtained from the wildform of the said Primulaceae organism. In doing this, for example,certain amino acids may be replaced by others having similarphysicochemical properties (space filling, basicity, hydrophobicity,etc.). For example, arginine residues are exchanged for lysine residues,valine residues for isoleucine residues or aspartic acid residues forglutamic acid residues. However, one or more amino acids may also beswapped in sequence, added or removed, or a plurality of these measuresmay be combined with one another.

By derivatives is also meant functional equivalents which in particularalso contain natural or artificial mutations of an originally isolatedsequence encoding Δ6-desaturase which continue to exhibit the desiredfunction, that is the enzymatic activity and substrate selectivitythereof is not substantially reduced. Mutations comprise substitutions,additions, deletions, exchanges or insertions of one or more nucleotideresidues. Thus, for example, the present invention also encompassesthose nucleotide sequences which are obtained by modification of theΔ6-desaturase nucleotide sequence. The aim of such a modification maybe, e.g., to farther bound the encoding sequence contained therein oralso, e.g. to insert further restriction enzyme interfaces.

Functional equivalents also include those variants whose function bycomparison with the initial gene or gene fragment is weakened (=notsubstantially reduced) or reinforced (=enzyme activity higher than theactivity of the initial enzyme, that is activity is higher than 100%,preferably higher than 110%, particularly preferably higher than 130%).

At the same time the nucleic acid sequence may, for example,advantageously be a DNA or cDNA sequence. Suitable encoding sequencesfor insertion into an expression cassette according to the inventioninclude by way of example those which encode a Δ6-desaturase with thesequences described above and lend the host the ability to overproducefatty acids, oils or lipids having double bonds in the Δ6 position, itbeing advantageous when at the same time ω-3 fatty acids having at leastfour double bonds are produced. These sequences may be of homologous orheterologous origin.

By the expression cassette (=nucleic acid construct or fragment or geneconstruct) according to the invention is meant the sequences specifiedin SEQ ID NO: 1 and SEQ ID NO:3 which result from the genetic codeand/or functional or nonfunctional derivatives thereof which arefunctionally linked with one or more regulation signals advantageouslyto increase the gene expression and which control the expression of theencoding sequence in the host cell. These regulatory sequences shouldallow the selective expression of the genes and the protein expression.Depending on the host organism this may mean, for example, that the geneis expressed and/or overexpressed only after induction or that it isexpressed and/or overexpressed immediately. Examples of these regulatorysequences are sequences to which inductors or repressors bind and inthis way regulate the expression of the nucleic acid. In addition tothese new regulation sequences or instead of these sequences the naturalregulation of these sequences ahead of the actual structural genes maystill be present and optionally have been genetically modified so thatnatural regulation was switched off and the expression of the genesincreased. However, the gene construct can also be built up more simply,that is no additional regulation signals have been inserted ahead of thenucleic acid sequence or derivatives thereof and the natural promoterwith its regulation has not been removed. Instead of this the naturalregulation sequence was mutated in such a way that no further regulationensues and/or the gene expression is heightened. These modifiedpromoters in the form of part sequences (=promoter containing parts ofthe nucleic acid sequences according to the invention) can also bebrought on their own ahead of the natural gene to increase the activity.In addition, the gene construct may advantageously also contain one ormore so-called enhancer sequences functionally linked to the promoterwhich allow enhanced expression of the nucleic acid sequence. At the 3′end of the DNA sequences additional advantageous sequences may also beinserted, such as further regulatory elements or terminators. TheΔ6-desaturase gene may be present in one or more copies in theexpression cassette (=gene construct).

As described above, the regulatory sequences or factors cal preferablypositively influence and so increase the gene expression of theintroduced genes. Thus, reinforcement of the regulatory elementsadvantageously on the transcription level may be effected by usingpowerful transcription signals such as promoters and/or enhancers.However, in addition reinforcement of translation is also possible, forexample by improving the stability of the mRNA.

Suitable promoters in the expression cassette are in principle allpromoters which can control the expression of foreign genes in organismssuch as microorganisms like protozoa such as ciliates, algae such asgreen, brown, red or blue algae, bacteria such as gram-positive orgram-negative bacteria, yeasts such as Saccharomyces, Pichia orSchizosaccharomyces or fungi such as Mortierella, Traustochytrium orSchizochytrium, advantageously in plants or fungi. Use is preferablymade in particular of plant promoters or promoters derived from a plantvirus. Advantageous regulation sequences for the method according to theinvention are found for example in promoters such as cos, tac, trp, tet,trp-tet, lpp, lac, lpp-lac, lacI^(q)-, T7, T5, T3, gal, trc, ara, SP6,λ-P_(R) or in λ-P_(L) promoters which are employed advantageously ingram-negative bacteria. Other advantageous regulation sequences arefound, for example, in the gram-positive promoters amy and SPO2, in theyeast or fungal promoters ADC1, MFα, AC, P-60, CYC1, GAPDH, TEF, rp28,ADH or in the plant promoters CaMV/35S [Franck et al., Cell 21 (1980)285-294], SSU, OCS, lib4, STLS1, B33, nos (=Nopalin Synthase Promoter)or in the ubiquintin promoter. The expression cassette may also containa chemically inducible promoter by means of which the expression of theexogenous Δ6-desaturase gene in the organisms can be controlledadvantageously in the plants at a particular time. Advantageous plantpromoters of this type are by way of example the PRP1 promoter [Ward etal., Plant. Mol. Biol. 22 (1993), 361-366], a promoter inducible bybenzenesulfonamide (EP 388186), a promoter inducible by tetracycline(Gatz et al., (1992) Plant J. 2, 397-404), a promoter inducible bysalicylic acid (WO 95/19443), a promoter inducible by abscisic acid(EP335528) and a promoter inducible by ethanol or cyclohexanone(WO93/21334). Other examples of plant promoters which can advantageouslybe used are the promoter of cytosolic FBPase from potato, the ST-LSIpromoter from potato (Stockhaus et al., EMBO J. 8 (1989) 2445-245), thepromoter of phosphoribosyl pyrophosphate amidotransferase from Glycinemax (see also gene bank accession number U87999) or a nodiene-specificpromoter as described in EP 249676. Particularly advantageous are thoseplant promoters which ensure expression in tissues or plant parts/organsin which fatty acid biosynthesis or the precursor stages thereof occurs,as in endosperm or in the developing embryo for example. Particularlynoteworthy are advantageous promoters which ensure seed-specificexpression such as by way of example the USP promoter or derivativesthereof, the LEB4 promoter, the phaseolin promoter or the napinpromoter. The particularly advantageous USP promoter cited according tothe invention or its derivatives mediate very early gene expression inseed development (Baeumlein et al., Mol Gen Genet, 1991, 225 (3):459-67). Other advantageous seed-specific promoters which may be usedfor monocotylodonous or dicotylodonous plants are the promoters suitablefor dicotylodons such as napin gene promoters, likewise cited by way ofexample, from oilseed rape (U.S. Pat. No. 5,608,152), the oleosinpromoter from Arabidopsis (WO 98/45461), the phaseolin promoter fromPhaseolus vulgaris (U.S. Pat. No. 5,504,200), the Bce4 promoter fromBrassica (WO 91/13980) or the leguminous B4 promoter (LeB4, Baeumlein etal., Plant J., 2, 2, 1992: 233-239) or promoters suitable formonocotylodons such as the promoters of the lpt2 or lpt1 gene in barley(WO 95/15389 and WO 95/23230) or the promoters of the barley hordeinegene, the rice glutelin gene, the rice oryzin gene, the rice prolamingene, the wheat gliadin gene, the white glutelin gene, the corn zeingene, the oats glutelin gene, the sorghum kasirin gene or the ryesecalin gene which are described in WO99/16890.

Furthermore, particularly preferred are those promoters which ensure theexpression in tissues or plant parts in which, for example, thebiosynthesis of fatty acids, oils and lipids or the precursor stagesthereof takes place. Particularly noteworthy are promoters which ensurea seed-specific expression. Noteworthy are the promoter of the napingene from oilseed rape (U.S. Pat. No. 5,608,152), the USP promoter fromVicia faba (USP=unknown seed protein, Baeumlein et al., Mol Gen Genet,1991, 225 (3): 459-67), the promoter of the oleosin gene fromArabidopsis (WO98/45461), the phaseolin promoter (U.S. Pat. No.5,504,200) or the promoter of the legumin B4 gene (LeB4; Baeumlein etal., 1992, Plant Journal, 2 (2): 233-9). Other promoters to be mentionedare that of the lpt2 or lpt1 gene from barley (WO95/15389 andWO95/23230) which mediate seed-specific expression in monocotyledonousplants.

As described above, the expression construct (=gene construct, nucleicacid construct) may contain yet other genes which are to be introducedinto the organisms. These genes can be subject to separate regulation orbe subject to the same regulation region as the Δ6-desaturase gene.These genes are by way of example other biosynthesis genes,advantageously for fatty acid biosynthesis, which allow increasedsynthesis. Examples which may be mentioned are the genes for Δ15, Δ12-,Δ9-, Δ6-, Δ5-, Δ4-desaturase, β-ketoacyl reductases, β-ketoacylsynthases, elongases or the various hydroxylases and acyl-ACPthioesterases. The desaturase genes are advantageously used in thenucleic acid construct.

In principle all natural promoters with their regulation sequences canbe used like those named above for the expression cassette according tothe invention and the method according to the invention. Over and abovethis, synthetic promoters may also advantageously be used.

In the preparation of an expression cassette various DNA fragments canbe manipulated in order to obtain a nucleotide sequence which usefullyreads in the correct direction and is equipped with a correct readingraster. To connect the DNA fragments (=nucleic acids according to theinvention) to one another adaptors or linkers may be attached to thefragments.

The promoter and the terminator regions can usefully be provided in thetranscription direction with a linker or polylinker containing one ormore restriction points for the insertion of this sequence. Generally,the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6, restrictionpoints. In general the size of the linker inside the regulatory regionis less than 100 bp, frequently less than 60 bp, but at least 5 bp. Thepromoter may be both native or homologous as well as foreign orheterologous to the host organism, for example to the host plant. In the5′-3′ transcription direction the expression cassette contains thepromoter, a DNA sequence which encodes a Δ6-desaturase gene and a regionfor transcription termination. Different termination regions can beexchanged for one another in any desired fashion.

Furthermore, manipulations which provide suitable restriction interfacesor which remove excess DNA or restriction interfaces can be employed.Where insertions, deletions or substitutions, such as transitions andtransversions, come into consideration, in vitro mutagenesis, primerrepair, restriction or ligation may be used. In suitable manipulationssuch as restriction, chewing back or filling of overhangs for blunt endscomplementary ends of the fragments can be provided for the ligation.

For an advantageous high expression the attachment of the specific ERretention signal SEKDEL inter alia can be of importance (Schouten, A. etal., Plant Mol. Biol. 30 (1996), 781-792). In this way the averageexpression level is tripled or even quadrupled. Other retention signalswhich occur naturally in plant and animal proteins located in the ER mayalso be employed for the construction of the cassette.

Preferred polyadenylation signals are plant polyadenylation signals,preferably those which substantially correspond to T-DNA polyadenylationsignals from Agrobacterium tumefaciens, in particular gene 3 of theT-DNA (octopin synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBOJ. 3 (1984), 835 et seq.) or corresponding functional equivalents.

An expression cassette is produced by fusion of a suitable promoterwith, a suitable Δ6-desaturase DNA sequence together with apolyadenylation signal by common recombination and cloning techniques asdescribed, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989) as well as in T. J. Silhavy, M. L.Berman and L. W. Enquist, Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M.et al., Current Protocols in Molecular Biology, Greene Publishing Assoc.and Wiley-Interscience (1987).

In the preparation of an expression cassette various DNA fragments canbe manipulated to produce a nucleotide sequence which usefully reads inthe correct direction and is equipped with a correct reading raster.Adapters or linkers can be attached to the fragments for joining the DNAfragments.

The promoter and the terminator regions can usefully be provided in thetranscription direction with a linker or polylinker containing one ormore restriction points for the insertion of this sequence. Generally,the linker has 1 to 10, mostly 1 to 8, preferably 2 to 6, restrictionpoints. In general the size of the linker inside the regulatory regionis less than 100 bp, frequently less than 60 bp, but at least 5 bp. Thepromoter may be both native or homologous as well as foreign orheterologous to the host organism, for example to the host plant. In the5′-3′ transcription direction the expression cassette contains thepromoter, a DNA sequence which encodes a Δ6-desaturase gene and a regionfor transcription termination. Different termination regions can beexchanged for one another in any desired fashion.

In the preparation of an expression cassette various DNA fragments canbe manipulated to produce a nucleotide sequence which usefully reads Inthe correct direction and is equipped with a correct reading raster.Adapters or linkers can be attached to the fragments for joining the DNAfragments.

The DNA sequences encoding two Δ6-desaturases from Muscariodides vialiiand Aleuritia farinosa contain all the sequence characteristics neededto achieve correct localization of the site of fatty acid, lipid or oilbiosynthesis. Accordingly, no further targeting sequences are needed perse. However, such a localization may be desirable and advantageous andhence artificially modified or reinforced so that such fusion constructsare also a preferred advantageous embodiment of the invention.

Particularly preferred are sequences which ensure targeting in plastids.Under certain circumstances targeting into other compartments (reportedin: Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423) may also bedesirable, e.g. into vacuoles, the mitochondrium, the endoplasmicreticulum (ER), peroxisomes, lipid structures or due to lack ofcorresponding operative sequences retention in the compartment oforigin, the cytosol.

Advantageously, the nucleic acid sequences according to the invention orthe gene construct together with at least one reporter gene are clonedinto an expression cassette which is introduced into the organism via avector or directly into the genome. This reporter gene should allow easydetection via a growth, fluorescence, chemical, bioluminescence orresistance assay or via a photometric measurement. Examples of reportergenes which may be mentioned are antibiotic- or herbicide-resistancegenes, hydrolase genes, fluorescence protein genes, bioluminescencegenes, sugar or nucleotide metabolic genes or biosynthesis genes such asthe Ura3 gene, the Ilv2 gene, the luciferase gene, the β-galactosidasegene, the gfp gene, the 2-desoxyglucose-6-phosphate phosphatase gene,the β-glucuronidase gene, β-lactamase gene, the neomycinphosphotransferase gene, the hygromycin phosphotransferase gene or theBASTA (=gluphosinate-resistance) gene. These genes permit easymeasurement and quantification of the transcription activity and henceof the expression of the genes. In this way genome positions may beidentified which exhibit differing productivity.

In a preferred embodiment an expression cassette comprises upstream,i.e. at the 5′ end of the encoding sequence, a promoter and downstream,i.e. at the 3′ end, a polyadenylation signal and optionally otherregulatory elements which are operably linked to the interveningencoding sequence for Δ6-desaturase and/or Δ6-desaturase DNA sequence.By an operable linkage is meant the sequential arrangement of promoter,encoding sequence, terminator and optionally other regulatory elementsin such a way that each of the regulatory elements can fulfill itsfunction in the expression of the encoding sequence in due manner. Thesequences preferred for operable linkage are targeting sequences forensuring subcellular localization in plastids. However, targetingsequences for ensuring subcellular localization in the mitochondrium, inthe endoplasmic reticulum (=ER), in the nucleus, in oil corpuscles orother compartments may also be employed as well as translation promoterssuch as the 5′ lead sequence in tobacco mosaic virus (Gallie et al.,Nucl. Acids Res. 15 (1987), 8693-8711).

An expression cassette may, for example, contain a constitutive promoteror a tissue-specific promoter (preferably the USP or napin promoter) thegene to be expressed and the ER retention signal. For the ER retentionsignal the KDEL amino acid sequence (lysine, aspartic acid, glutamicacid, leucine) or the KKX amino acid sequence (lysine-lysine-X-stop,wherein X means every other known amino acid) is preferably employed.

For expression in a prokaryotic or eukaryotic host organism, for examplea microorganism such as a fungus or a plant the expression cassette isadvantageously inserted into a vector such as by way of example aplasmid, a phage or other DNA which allows optimum expression of thegenes in the host organism. Examples of suitable plasmids are: in E.coli pLG338, pACYC184, pBR series such as e.g. pBR322, pUC series suchas pUC18 or pUC19, M113mp series, pKC30, pRep4, pHS1, pHS2, pPLc236,pMBL24, pLG200, pUR290, pIN-III¹¹³-B1, λgt11 or pBdCI; in StreptomycespIJ101, pIJ364, pIJ702 or pIJ361; in Bacillus pUB110, pC194 or pBD214;in Corynebacterium pSA77 or pAJ667; in fungi pALS1, pIL2 or pBB116;other advantageous fungal vectors are described by Romanos, M. A. etal., [(1992) “Foreign gene expression in yeast: a review”, Yeast 8:423-488] and by van den Hondel, C. A. M. J. J. et al. [(1991)“Heterologous gene expression in filamentous fungi” as well as in MoreGene Manipulations in Fungi [J. W. Bennet & L. L. Lasure, eds., pp.396-428: Academic Press: San Diego] and in “Gene transfer systems andvector development for filamentous fungi” [van den Hondel, C. A. M. J.J. & Punt, P. J. (1991) in: Applied Molecular Genetics of Fungi,Peberdy, J. F. et al., eds., pp. 1-28, Cambridge University Press:Cambridge]. Examples of advantageous yeast promoters are 2∝M, pAG-1,YEp6, YEp13 or pEMBLYe23. Examples of algal or plant promoters arepLGV23, pGHlac⁺, pBIN19, pAK2004, pVKH or pDH51 (see Schmidt, R. andWillmitzer, L., 1988). The vectors identified above or derivatives ofthe vectors identified above are a small selection of the possibleplasmids. Further plasmids are well known to those skilled in the artand may be found, for example, in the book Cloning Vectors (Eds. PouwelsP. H. et al. Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444904018). Suitable plant vectors are described inter alia in “Methods inPlant Molecular Biology and Biotechnology” (CRC Press), Ch. 6/7, pp.71-119. Advantageous vectors are known as shuttle vectors or binaryvectors which replicate in E. coli and Agrobacterium.

By vectors is meant with the exception of plasmids all other vectorsknown to those skilled in the art such as by way of example phages,viruses such as SV40, CMV, baculovirus, adenovirus, transposons, ISelements, phasmids, phagemids, cosmids, linear or circular DNA. Thosevectors can be replicated autonomously in the host organism or bechromosomally replicated, chromosomal replication being preferred.

In a further embodiment of the vector the expression cassette accordingto the invention may also advantageously be introduced into theorganisms in the form of a linear DNA and be integrated into the genomeof the host organism by way of heterologous or homologous recombination.This linear DNA may be composed of a linearized plasmid or only of theexpression cassette as vector or the nucleic acid sequences according tothe invention.

In a further advantageous embodiment the nucleic acid sequence accordingto the invention can also be introduced into an organism on its own.

If in addition to the nucleic acid sequence according to the inventionfurther genes are to be introduced into the organism, all together witha reporter gene in a single vector or each single gene with a reportergene in a vector in each case can be introduced into the organism,whereby the different vectors can be introduced simultaneously orsuccessively.

The vector advantageously contains at least one copy of the nucleic acidsequences according to the invention and/or the expression cassette(=gene construct) according to the invention.

By way of example the plant expression cassette can be installed in thepRT transformation vector ((a) Toepfer et al., 1993, Methods Enzymol.,217: 66-78; (b) Toepfer et al. 1987, Nucl. Acids. Res. 15: 5890 ff.).

Alternatively, a recombinant vector (=expression vector) can also betranscribed and translated in vitro, e.g. by using the T7 promoter andthe T7 RNA polymerase.

Expression vectors employed in prokaryotes frequently make use ofinducible systems with and without fusion proteins or fusionoligopeptides, wherein these fusions can ensue in both N-terminal andC-terminal manner or in other useful domains of a protein. Such fusionvectors usually have the following purposes: i.) to increase the RNAexpression rate; ii.) to increase the achievable protein synthesis rate;iii.) to increase the solubility of the protein; iv.) or to simplifypurification by means of a binding sequence usable for affinitychromatography. Proteolytic cleavage points are also frequentlyintroduced via fusion proteins which allows cleavage of a portion of thefusion protein and purification. Such recognition sequences forproteases are recognized, e.g. factor Xa, thrombin and enterokinase.

Typical advantageous fusion and expression vectors are pGEX [PharmaciaBiotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67: 31-40],pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which contains glutathione S-transferase (GST),maltose binding protein or protein A.

Other examples of E. coli expression vectors are pTrc [Amann et al.,(1988) Gene 69:301-315] and pET vectors [Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89; Stratagene, Amsterdam, The Netherlands].

Other advantageous vectors for use in yeast are pYepSec1 (Baldari, etal., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYESderivatives (Invitrogen Corporation, San Diego, Calif.). Vectors for usein filamentous fungi are described in: van den Hondel, C. A. M. J. J. &Punt, P. J. (1991) “Gene transfer systems and vector development forfilamentous fungi”, in: Applied Molecular Genetics of Fungi, J. F.Peberdy, et al., eds., pp. 1-28, Cambridge University Press: Cambridge.

Alternatively, insect cell expression vectors can also be advantageouslyutilized, e.g. for expression in Sf9 cells. These are e.g. the vectorsof the pAc series (Smith et al. (1983) Mol. Cell. Biol. 3:2156-2165) andthe pVL series (Lucklow and Sunmmers (1989) Virology 170:31-39).

Furthermore, plant cells or algal cells can advantageously be used forgene expression. Examples of plant expression vectors may be found inBecker, D., et al. (1992) “New plant binary vectors with selectablemarkers located proximal to the left border”, Plant Mol. Biol. 20:1195-1197 or in Bevan, M. W. (1984) “Binary Agrobacterium vectors forplant transformation”, Nucl. Acid Res. 12: 8711-8721.

Furthermore, the nucleic acid sequences according to the invention maybe expressed in mammalian cells, advantageously in nonhuman mammaliancells. Examples of corresponding expression vectors are pCDM8 and pMT2PCreferred to in: Seed, B. (1987) Nature 329:840 or Kaufman et al. (1987)EMBO J. 6: 187-195). At the same time promoters preferred for use are ofviral origin, such as by way of example promoters of polyoma, adenovirus2, cytomegalovirus or simian virus 40. Other prokaryotic and eukaryoticexpression systems are referred to in chapters 16 and 17 of Sambrook etal., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

The introduction of the nucleic acids according to the invention, theexpression cassette or the vector into organisms, plants for example,can in principle be done by all of the methods known to those skilled inthe art. The introduction of the nucleic acid sequences gives rise torecombinant or transgenic organisms.

In the case of microorganisms, those skilled in the art can findappropriate methods in the textbooks by Sambrook, J. et al. (1989)Molecular cloning: A laboratory manual, Cold Spring Harbor LaboratoryPress, by F. M. Ausubel et al. (1994) Current protocols in molecularbiology, John Wiley and Sons, by D. M. Glover et al., DNA Cloning Vol.1, (1995), IRL Press (ISBN 019-963476-9), by Kaiser et al. (1994)Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press orGuthrie et al. Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, 1994, Academic Press.

The transfer of foreign genes into the genome of a plant is calledtransformation. In doing this the methods described for thetransformation and regeneration of plants from plant tissues or plantcells are utilized for transient or stable transformation. Suitablemethods are protoplast transformation by poly(ethylene glycol)-inducedDNA uptake, the “biolistic” method using the gene cannon—referred to asthe particle bombardment method, electroporation, the incubation of dryembryos in DNA solution, microinjection and gene transfer mediated byAgrobacterium. Said methods are described by way of example in B. Jeneset al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press(1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec.Biol. 42 (1991) 205-225). The construct to be expressed is preferablycloned into a vector which is suitable for transforming Agrobacteriumtumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12(1984) 8711). Agrobacteria transformed by such a vector can then be usedin known manner for the transformation of plants, in particular of cropplants such as by way of example tobacco plants, for example by bathingbruised leaves or chopped leaves in an agrobacterial solution and thenculturing them in suitable media. The transformation of plants by meansof Agrobacterium tumefaciens is described, for example, by Höfgen andWillmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter aliafrom F. F. White, Vectors for Gene Transfer in Higher Plants; inTransgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kungand R. Wu, Academic Press, 1993, pp. 15-38.

Agrobacteria transformed by an expression vector according to theinvention may likewise be used in known manner for the transformation ofplants such as test plants like Arabidopsis or crop plants such ascereal crops, corm, oats, rye, barley, wheat, soybean, rice, cotton,sugar beet, canola, sunflower, flax, hemp, potatoes, tobacco, tomatoes,carrots, paprika, oilseed rape, tapioca, cassava, arrowroot, targets,alfalfa, lettuce and the various tree, nut and vine species, inparticular of oil-containing crop plants such as soybean, peanut, castoroil plant, sunflower, corn, cotton, flax, oilseed rape, coconut, oilpalm, safflower (Carthamus tinctorius) or cocoa bean, e.g. by bathingbruised leaves or chopped leaves in an agrobacterial solution and thenculturing them in suitable media. For the production of PUFAs, forexample stearidonic acid, eicosapentaenoic acid and docosahexaenoicacid, borage or Primulaccae are advantageously suitable.

The genetically modified plant cells may be regenerated by all of themethods known to those skilled in the art. Appropriate methods can befound in the publications referred to above by S. D. Kung and R. Wu,Potrykus or Höfgen and Willmitzer.

Accordingly, a further aspect of the invention relates to transgenicorganisms transformed by at least one nucleic acid sequence, expressioncassette or vector according to the invention as well as cells, cellcultures, tissue, parts—such as, for example, leaves, roots, etc. in thecase of plant organisms—or reproductive material derived from suchorganisms. The terms “host organism”, “host cell”, “recombinant (host)organism” and “transgenic (host) cell” are used here interchangeably. Ofcourse these terms relate not only to the particular host organism orthe particular target cell but also to the descendants or potentialdescendants of these organisms or cells. Since, due to mutation orenvironmental effects certain modifications may arise in successivegenerations, these descendants need not necessarily be identical withthe parental cell but nevertheless are still encompassed by the term asused here.

For the purposes of the invention “transgenic” or “recombinant” meanswith regard for example to a nucleic acid sequence, an expressioncassette (=gene construct) or a vector containing the nucleic acidsequence according to the invention or an organism transformed by thenucleic acid sequences, expression cassette or vector according to theinvention all those constructions produced by genetic engineeringmethods in which either

a) the nucleic acid sequence according to the invention or

b) a genetic control sequence functionally linked to the nucleic acidsequence according to the invention, for example a promoter, or

c) (a) and (b)

are not found in their natural, genetic environment or have beenmodified by genetic engineering methods, wherein the modification may byway of example be a substitution, addition, deletion, inversion orinsertion of one or more nucleotide residues. Natural geneticenvironment means the natural genomic or chromosomal locus in theorganism of origin or presence in a genomic library. In the case of agenomic library the natural genetic environment of the nucleic acidsequence is preferably retained at least in part. The environmentborders the nucleic acid sequence at least on one side and has asequence length of at least 50 bp, preferably at least 500 bp,particularly preferably at least 1,000 bp, most particularly preferablyat least 5,000 bp. A naturally occurring expression cassette—for examplethe naturally occurring combination of the natural promoter of thenucleic acid sequence according to the invention with the correspondingPSE gene—turns into a transgenic expression cassette when the latter ismodified by unnatural, synthetic (“artificial”) methods such as by wayof example a mutagenation. Appropriate methods are described by way ofexample in U.S. Pat. No. 5,565,350 or WO 00/15815.

Suitable organisms or host organisms for the nucleic acid, expressioncassette or vector according to the invention are advantageously inprinciple all organisms which are able to synthesize fatty acids,especially unsaturated fatty acids or are suitable for the expression ofrecombinant genes. Examples which may be mentioned are plants such asArabidopsis, Asteraceae such as Calendula or crop plants such assoybean, peanut, castor oil plant, sunflower, corn, cotton, flax,oilseed rape, coconut, oil palm, safflower (Carthamus tinctorius) orcocoa bean, microorganisms such as fungi, for example the genusMortierella, Saprolegnia or Pythium, bacteria such as the genusEscherichia, yeasts such as the genus Saccharomyces, cyanobacteria,ciliates, algae or protozoa such as dinoflagellates likeCrypthecodinium. Preference is given to organisms which can naturallysynthesize oils in relatively large quantities such as fungi likeMortierella alpina, Pythium insidiosum or plants such as soybean,oilseed rape, coconut, oil palm, safflower, flax, castor oil plant,Calendula, peanut, cocoa bean or sunflower, or yeasts such asSaccharomyces cerevisiae and particular preference is given to soybean,flax, oilseed rape, sunflower, Calendula, Mortierella or Saccharomycescerevisiae. In principle, apart from the transgenic organisms identifiedabove, transgenic animals, advantageously nonhuman animals, aresuitable, for example C. elegans.

Further useful host cells arm identified in: Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990).

Usable expression strains, e.g. those exhibiting a relatively lowprotease activity, are described in: Gottesman, S., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 119-128.

A further object of the invention relates to the use of an expressioncassette containing DNA sequences encoding a Δ6-desaturase gene or DNAsequences hybridizing therewith for the transformation of plant cells,tissues or parts of plants. The aim of use is to increase the content offatty acids, oils or lipids having an increased content of double bondsin the Δ6 position.

In doing so, depending on the choice of promoter, the Δ6-desaturase genecan be expressed specifically in the leaves, in the seeds, the nodules,in roots, in the stem or other parts of the plant. Those transgenicplants overproducing fatty acids, oils or lipids having Δ6 double bonds,the reproductive material thereof, together with the plant cells,tissues or parts thereof are a further object of the present invention.A preferred object according to the invention comprises transgenicplants containing a functional or nonfunctional (=antisense DNA orenzymatically inactive enzyme) nucleic acid sequence or a functional ornonfunctional expression cassette according to the invention.

The expression cassette or the nucleic acid sequences according to theinvention containing a Δ6-desaturase gene sequence can, moreover, alsobe employed for the transformation of the organisms identified by way ofexample above such as bacteria, cyanobacteria, yeasts, filamentousfungi, ciliates and algae with the objective of increasing the contentof fatty acids, oils or lipids possessing Δ6 double bonds.

Within the framework of the present invention, increasing the content offatty acids, oils or lipids possessing Δ6 double bonds means, forexample, the artificially acquired trait of increased biosyntheticperformance due to functional overexpression of the Δ6-desaturase genein the organisms according to the invention, advantageously in thetransgenic plants according to the invention, by comparison with thenongenetically modified initial plants at least for the duration of atleast one plant generation.

The preferred locus of biosynthesis, of fatty acids, oils or lipids forexample, is generally the seed or cell layers of the seed so that aseed-specific expression of the Δ6-desaturase gene is appropriate. Itis, however, obvious that the biosynthesis of fatty acids, oils orlipids need not be limited to the seed tissue but rather can also occurin tissue-specific manner in all other parts of the plant—in epidermiscells or in the nodules for example.

A constitutive expression of the exogenous Δ6-desaturase gene is,moreover, advantageous. On the other hand, however, an inducibleexpression may also appear desirable.

The efficiency of the expression of the Δ6-desaturase gene can bedetermined, for example, in vitro by shoot meristem propagation. Inaddition, an expression of the Δ6-desaturase gene modified in nature andlevel and its effect on fatty acid, oil or lipid biosynthesisperformance can be tested on test plants in greenhouse trials.

An additional object of the invention comprises transgenic organismssuch as transgenic plants transformed by an expression cassettecontaining a Δ6-desaturase gene sequence according to the invention orDNA sequences hybridizing therewith, as well as transgenic cells,tissue, parts and reproduction material of such plants. Particularpreference is given in this case to transgenic crop plants such as byway of example barley, wheat, rye, oats, corn, soybean, rice, cotton,sugar beet, oilseed rape and canola, sunflower, flax, hemp, thistle,potatoes, tobacco, tomatoes, oilseed rape, tapioca, cassava, arrowroot,alfalfa, lettuce and the various tree, nut and vine species.

For the purposes of the invention plants are mono- and dikotylodonousplants, mosses or algae.

A further refinement according to the invention are transgenic plants asdescribed above which contain a functional or nonfunctional nucleic acidsequence according to the invention or a functional or nonfunctionalexpression cassette according to the invention. By nonfunctional ismeant that an enzymatically active protein is no longer synthesized.Moreover, by nonfunctional nucleic acids or nucleic acid constructs isalso means what is known as an antisense DNA which results in transgenicplants which show a reduction in enzymatic activity or no enzymaticactivity. With the aid of the antisense technique, especially when thenucleic acid sequence is combined in the antisense DNA with other fattyacid synthesis genes, it is possible to synthesize triglycerides havingan increased content of saturated fatty acids or saturated fatty acids.Furthermore, by means of what is known as coexpression or by means ofthe RNAi technique transgenic plants can be manipulated in such a waythat no or reduced enzymatic activity is produced in the plants. Bytransgenic plants is meant single plant cells and cultures thereof onfixed media or in liquid culture, parts of plants and entire plants.

Other objects of the invention are:

-   -   A method for the transformation of a plant comprising the        introduction of expression cassettes according to the invention        containing a Δ6-desaturase gene sequence derived from        Primulaceae or DNA sequences hybridizing therewith into a plant        cell, into callus tissue, an entire plant or protoplasts of        plants.    -   A method for producing PUFAs, wherein the method comprises the        growing of a transgenic organism comprising a nucleic acid as        claimed in claims 1 to 4, a gene construct as claimed in claim 6        or a vector as claimed in claim 7 encoding a Δ6-desaturase which        specifically desaturates ω-3 fatty acids, and wherein due to the        activity of the Δ6-desaturase PUFAs are formed in the organism        which exhibit an increased content of ω-3 fatty acids. In this        method ω-3 fatty acids such as stearidonic acid,        eicosapentaenoic acid or docosahexaenoic acid are advantageously        produced.

Use of a Δ6-desaturase DNA gene sequence or DNA sequences hybridizingtherewith for the production of plants having an increased content offatty acids, oils or lipids having Δ6 double bonds due to the expressionof said Δ6-desaturase DNA sequence in plants.

Use of a Δ6-desaturase DNA gene sequence DNA sequences hybridizingtherewith for the production of plants having an increased content offatty acids, oils or lipids having Δ6 double bonds, particularly of ω-3fatty acids, due to the expression of said Δ6-desaturase DNA sequence inplants.

-   -   Proteins containing the amino acid sequences depicted in SEQ ID        NO: 2 or NO: 4.    -   Use of said proteins having the sequences SEQ ID NO: 2 or NO: 4        for producing unsaturated fatty acids.

A further object according to the invention is a method for producingunsaturated fatty acids comprising: introducing at least one saidnucleic acid sequence according to the invention or at least one nucleicacid construct according to the invention into a preferablyoil-producing plant; growing said organism; isolating oil contained insaid organism; and liberating the fatty acids present in said oil. Theseunsaturated fatty acids advantageously contain Δ6 double bonds. Thefatty acids may be liberated from the oils or lipids, for example bybasic hydrolysis, e.g. using NaOH or KOH.

A method for producing triglycerides having an increased content ofunsaturated fatty acids comprising: introducing at least one saidnucleic acid sequence according to the invention or at least oneexpression cassette according to the invention into an oil-producingorganism; growing said organism; and isolating oil contained in saidorganism; is also numbered among the objects of the invention.

A further object according to the invention is a method for producingtriglycerides having an increased content of unsaturated fatty acids byincubating triglycerides containing saturated or unsaturated orsaturated and unsaturated fatty acids with at least one of the proteinsencoded by the sequences SEQ ID NO: 2 or NO: 4. The method isadvantageously carried out in the presence of compounds which can takeup or release reduction equivalents. The fatty acids can then beliberated from the triglycerides.

A further object according to the invention of said method for producingtriglycerides having an increased content of saturated or unsaturatedfatty acids or saturated and unsaturated fatty acids advantageouslyhaving an increased content of unsaturated fatty acids is a methodwherein the fatty acids are liberated from the triglycerides with theaid of basic hydrolysis known to those skilled in the art or by means ofan enzyme such as a lipase.

The methods specified above advantageously allow the synthesis of fattyacids or triglycerides having an increased content of fatty acidscontaining Δ6 double bonds.

The methods identified above advantageously allow the synthesis of fattyacids or triglycerides having an increased content of fatty acidscontaining Δ6 double bonds, wherein the substrate used for the reactionof the Δ6-desaturase is preferably α-linolenic acid. In this way themethod identified above advantageously allows in particular thesynthesis of fatty acids derived from stearidonic acid (C_(18:4)^(Δ6,9,12,15)) such as by way of example eicosapentaenoic acid anddocosahexaenoic acid.

Using what is known as antisense technology, in one method fatty acidsor triglycerides having an increased content of saturated fatty acidscan also be produced.

Examples of organisms for the said methods which may be mentioned areplants such as Arabidopsis, Primulaceae, borage, barley, wheat, rye,oats, corn, soybean, rise, cotton, sugar beet, oilseed rape and canola,sunflower, flax, hemp, potatoes, tobacco, tomatoes, rape, tapioca,cassava, arrowroot, alfalfa, peanut, castor oil plant, coconut, oilpalm, safflower (Carthamus tinctorius) or cocoa bean, microorganismssuch as the fungi Mortierella, Saprolegnia or Pythium, bacteria such asthe genus Escherichia, cyanobacteria, yeasts such as the genusSaccharomyces, algae or protozoa such as dinoflagellates likeCrypthecodinium. Preference is given to organisms which can naturallysynthesize oils in relatively large quantities such as fungi likeMortierella alpina, Pythium insidiosum or plants such as soybean,oilseed rape, coconut, oil palm, safflower, castor oil plant, Calendula,peanut, cocoa bean or sunflower, or yeasts such as Saccharomycescerevisiae and particular preference is given to soybean, oilseed rape,sunflower, flax, Primulaceae, borage, Carthamus or Saccharomycescerevisiae.

Depending on the host organism, the organisms used in the methods aregrown or cultured in the manner known to those skilled in the art.Microorganisms are usually grown in a liquid medium containing a carbonsource, usually in the form of sugars, a nitrogen source, usually in theform of organic nitrogen sources such as yeast extract or salts such asammonium sulfate, trace elements such as iron, manganese or magnesiumsalts and optionally vitamins at temperatures of between 10_C and 60_Cwith exposure to gaseous oxygen. In doing so the pH of the nutrientliquid may be kept at a fixed value, that is during growth it is or isnot regulated. Growth can ensue in batch mode, semibatch mode orcontinuously. Nutrients can be provided at the start of fermentation orbe fed in semicontinuously or continuously.

After transformation plants are first of all regenerated as describedabove and then cultured or cultivated as normal.

After growth the lipids are isolated from the organisms in the usualway. For this purpose, after harvesting the organisms may first of allbe digested or used directly. The lipids are advantageously extractedusing suitable solvents such as apolar solvents like hexane or ethanol,isopropanol or mixtures such as hexane/isopropanol,phenol/chloroform/isoamyl alcohol at temperatures of between 0° C. and80° C., preferably between 20° C. and 50° C. The biomass is usuallyextracted with an excess of solvent, for example an excess of solvent tobiomass of 1:4. The solvent is then removed, for example bydistillation. Extraction can also be done using supercritical CO₂. Afterextraction the remaining biomass may be removed, for example byfiltration.

The crude oil isolated in this way can then be further purified, forexample by removing cloudiness by treatment with polar solvents such asacetone or chloroform and then filtration or centrifugation. Furtherpurification through columns is also possible.

In order to obtain the free acids from the triglycerides the latter aresaponified in the usual way.

A further object of the invention comprises fatty acids andtriglycerides having an increased content of unsaturated fatty acidsproduced by the methods identified above and use thereof for producingfoods, animal feeds, cosmetics or pharmaceuticals. For this purpose thelatter are added in customary quantities to the foods, the animal feed,the cosmetics or pharmaceuticals.

Said unsaturated fatty acids according to the invention as well astriglycerides having an increased content of unsaturated fatty acidsproduced by the methods identified above are the result of theexpression of the nucleic acids according to the invention in thevarious host organisms. This results overall in a modification of thecomposition of the compounds in the host cell containing unsaturatedfatty acids by comparison with the original starting host cells which donot contain the nucleic acids. These modifications are more marked inhost organisms, for example plant cells, which naturally do not containthe proteins or enzymes encoded by the nucleic acids than in hostorganisms which naturally do contain the proteins or enzymes encoded bythe nucleic acids. This gives rise to host organisms containing oils,lipids, phospholipids, sphingolipids, glycolipids, triacylglycerolsand/or free fatty acids having a higher content of PUFAs. For thepurposes of the invention, by an increased content is meant that thehost organisms contain at least 5%, advantageously at least 10%,preferably at least 20%, particularly preferably at least 30%, mostparticularly preferably at least 40% more polyunsaturated fatty acids bycomparison with the initial organism which does not contain the nucleicacids according to the invention. This is particularly the case forplants which do not naturally contain longer-chain polyunsaturated C₂₀or C₂₂ fatty acids such as DHA, EPA or ARA. Due to the expression of thenucleic acids novel lipid compositions are produced by said means thesebeing a further aspect of the invention.

The invention is explained in more detail by the following examples.

EXAMPLES Example 1 General Cloning Methods

The cloning methods, such as by way of example restriction cleavages,agarose gel electrophoresis, purification of DNA fragments, transfer ofnucleic acids to nitrocellulose and nylon membranes, linkage of DNAfragments, transformation of Escherichia coli cells, culture of bacteriaand sequence analysis of recombinant DNA, were carried out as describedin Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN0-87969-309-6).

Example 2 Sequence Analysis of Recombinant DNA

Sequencing of recombinant DNA molecules was done using a laserfluorescence DNA sequencer from the ABI company by the method of Sanger(Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467).Fragments resulting from a polymerase chain reaction were sequenced andchecked to prevent polymerase errors in the constructs to be expressed.

Example 3 Cloning of the Δ6-Desaturase from Muscariodides vialii (=SEQID NO: 3)

Total RNA from young Muscariodides vialii leaves was isolated with theaid of the RNAeasy kit from the Qiagen company (Valencia, Calif., USA).With the aid of oligo-dT-cellulose poly-A+ RNA (mRNA) was isolated fromthe total RNA (Sambrook et al., 1989). Using the Reverse TranscriptionSystem kit from Promega the RNA was reverse transcribed and thesynthesized cDNA was used for PCR amplification of the Δ6-desaturases.Degenerate primers were used for the amplification of the Δ6-desaturase.The nucleotide sequence was derived from the first and the thirdhistidine box motif of borage Δ6-desaturase (Syanova et al., 1997,WO9621022).

Primer 1: GGITGGHTIGGICAYGAYKYIKSICA (SEQ ID NO: 5) Primer 2:GGRAAIAGRTGRTGYTCDATYTG (SEQ ID NO: 6)

In the primers identified here and in the primer sequences set out belowthe symbols or letters in accordance with Wobble IUPAC-IUB have thefollowing meaning:

R=A/G; Y=C/T; M=A/C; K=G/T; S G/C; W=A/T; H=A/C/T; B=G/T/C; V=G/C/A;D=G/T/A and N=G/A/T/C.

PCR Protocol

Addition temperature: 1 min at 45° C.Denaturing temperature: 1 min at 94° C.Elongation temperature: 2 min at 72° C.Number of cycles: 35

The PCR mixture was separated on an agarose gel and a 660 bp fragmentwas isolated. The PCR fragment was cloned in the pGEM-T easy vector(Promega) and the insert was then sequenced.

The missing 5′ and 3′ region of the isolated gene fragment fromMuscariodides vialii was isolated with the aid of the Smart RACE cDNAkit (Clonetech) and then sequenced. Starting from 3′ and 5′ sequenceprimers were derived in order to isolate the complete clone. For thispurpose primers were derived from the DNA regions around the startmethionine and the stop codon. The PCR yielded a single band of theexpected size. The cDNA was again cloned in the pGEM T easy vector andthe now complete gene was sequenced.

Example 4 Cloning of the Δ6-Desaturase from Aleuritia farinosa (=SEQ IDNO: 1)

Total RNA from young Aleuritia farinose leaves was isolated with the aidof the RNAeasy kit from the Qiagen company (Valencia, Calif., USA). Withthe aid of oligo-dT-cellulose poly-A+ RNA (mRNA) was isolated from thetotal RNA (Sambrook et al., 1989). Using the Reverse TranscriptionSystem kit from Promega the RNA was reverse transcribed and thesynthesized cDNA was used for PCR amplification of the Δ6-desaturses.Degenerate primers were used for the amplification of the A6-desaturase. The nucleotide sequence was derived from the first and thethird histidine box motif of borage Δ6-desaturase (Syanova et al., 1997,WO9621022).

Primer 1: GGITGGHTIGGICAYGAYKYIKSICA (SEQ ID NO: 5) Primer 2:GGRAAIAGRTGRTGYTCDATYTG (SEQ ID NO: 6)

PCR Protocol

Addition temperature: 1 min at 45° C.Denaturing temperature: 1 min at 94° C.Elongation temperature: 2 min at 72° C.Number of cycles: 35

The PCR mixture was separated on an agarose gel and a 660 bp fragmentwas isolated. The PCR fragment was cloned in the pGEM-T easy vector(Promega) and the insert was then sequenced.

The missing 5′ and 3′ region of the isolated gene fragment fromAleuritia farinose was isolated with the aid of the Smart RACE cDNA kit(Clonetech) and then sequenced. Starting from 3′ and 5′ sequence primerswere derived in order to isolate the complete clone. For this purposeprimers were derived from the DNA regions around the start methionineand the stop codon. The PCR yielded a single band of the expected size.The cDNA was again cloned in the pGEM T easy vector and the now completegene was sequenced.

Example 5 Cloning of Expression Plasmids for Constitutive Expression inPlants

By means of appropriate primers at the 5′ and 3′ end of both newdesaturases a CLAI and a XbaI interface was introduced.

Primer design for the Δ6-desaturase from M. vialii:

atcgatatggctaacaaatctcccacc (ClaI) (SEQ ID NO: 7)tctagattagccgtgtgtgtggacggctt (XbaI) (SEQ ID NO: 8)

Primer design for the Δ6-desaturase from A. farinosa:

atcgatatggctaacaaatctcccacc (ClaI) (SEQ ID NO: 7)tctagatcacccgagagttttaagagct (XbaI) (SEQ ID NO: 9)

The PCR products were separated in agarose gel, digested with ClaI/XbaIand ligated into the appropriately cut vector pSLJ4K1. The resultantplasmids contain 35S promoter (cauliflower mosaic virus; Franck et al.(1980) Cell 21, 285), the Δ6-desaturase from Muscariodides vialii orAleuritia farinosa and the 35S terminator in the vector pSLJ4K1. Apartfrom said promoters or terminators all constitutive promoters or allplant virus promoters such as advantageously the nos promoter (Wilkinsonet al., Journal of Experimental Botany, 48, 1997: 307 et seq.) or theubiquintin promoter may be used. The promoters and terminatorsidentified in the description may also advantageously be used inprinciple for expression.

The constructs were used for the transformation of Arabidopsis thaliana,oilseed rape, tobacco and linseed.

Example 5 Cloning of Expression Plasmids for Seed-Specific Expression inPlants

For the transformation of plants a further transformation vector basedon pBin-USP containing the BamHI fragments of the Δ6-desaturases from M.vialii or A. farinosa was produced. The BamHI interfaces were, asdescribed in Example 4 [5? This is the second Example 5!], attached tothe start ATGs or stop codons with the aid of appropriate primers byPCR.

Primer Design for the Δ6-Desaturase from M. vialii:

ggatccatggctaacaaatctcccacc (SEQ ID NO: 10)ggatccttagccgtgtgtgtggacggctt (SEQ ID NO: 11)

Primer Design for the Δ6-Desaturase from A. farinosa:

ggatccatggctaacaaatctcccacc (SEQ ID NO: 10) ggatcctcacccgagagttttaagagct(SEQ ID NO: 12)

pBin-USP is a derivative of the plasmid pBin19 pBin-USP was producedfrom pBin19 by inserting a USP promoter as an EcoRI-BaMHI fragment intopBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711). Thepolyadenylation signal is that of gene 3 of the T-DNA of the Ti plasmidpTiACH5 (Gielen et al., (1984) EMBO J. 3, 835), whereby nucleotides11749-11939 were isolated as a PvuII-HindIII fragment and after additionof SphI linkers to the PvuII interface between the SpHI-HindIIIinterface of the vector were cloned. The USP promoter corresponds tonucleotides 1-684 (gene bank accession number X56240), wherein a part ofthe nonencoding region of the USP gene is contained in the promoter. Thepromoter fragment running to 684 base pairs was amplified by standardmethods by means of commercial T7 standard primer (Stratagene) and usinga synthesized primer through a PCR reaction. (Primer sequence:5′-GTCGACCCGCGGACTAGTGGGCCCTCTAGACCCGGGGGATCC GGATCTGCTGGCTATGAA-3′ (SEQID NO: 13)). The PCR fragment was recut using EcoRI/SalI and insertedinto the vector pBin19 with OCS terminator. The plasmid having thedesignation pBinUSP was obtained. The constructs were used fortransforming Arabidopsis thaliana, oilseed rape, tobacco and linseed.

Example 6 Production of Transgenic Plants

a) Production of transgenic plants (modified in accordance with Moloneyet al., 1992, Plant Cell Reports, 8:238-242)

To produce transgenic oilseed rape plants binary vectors inAgrobacterium tumefaciens C58C1-pGV2260 or Escherichia coli were used(Deblaere et al, 1984, Nucl. Acids. Res. 13, 4777-4788). Fortransforming oilseed rape plants (var. Drakkar, NPZ NordeutschePflanzenzucht, Hohenlieth. Germany) a 1:50 dilution of an overnightculture of a positively transformed agrobacteria colony inMurashige-Skoog medium (Murashige and Skoog 1962 Physiol. Plant. 15,473) containing 3% of saccharose (3MS medium) was used. Petioles orhypocotyledons of freshly germinated sterile rape plants (approx. 1 cm²each) were incubated in a Petri dish with a 1:50 agrobacteria dilutionfor 5-10 minutes. This was followed by 3-day concubation in darkness at25_C on 3MS medium containing 0.8% of Bacto-Agar. After three days,culturing was continued with 16 hours of light/8 hours of darkness andin a weekly cycle on MS medium containing 500 mg/l of Claforan (sodiumcefotaxime), 50 mg/l of kanamycin, 20 microM of benzylaminopurine (BAP)and 1.6 g/l of glucose. Growing shoots were transferred onto MS mediumcontaining 2% of saccharose, 250 mg/l of Claforan and 0.8% ofBacto-Agar. If after three weeks no roots had formed 2-indolylbutyricacid was added to the medium as a growth hormone for rooting purposes.

Regenerated shoots were obtained on 2MS medium using kanamycin andClaforan, transferred into soil after rooting and after culturing grownfor two weeks in a climate-controlled chamber, brought to blossom andafter harvesting of ripe seed investigated for Δ6-desaturase expressionby means of lipid analyses. Lines having increased contents of doublebonds at the Δ6 position were identified. In the stably transformtransgenic lines functionally expressing the transgene it was found thatthere is an increased content of double bonds at the Δ6 position bycomparison with untransformed control plants.

b) Transgenic flax plants may be produced, for example by the by themethod Bell et al., 1999, In Vitro Cell. Dev. Biol. Plant.35(6):456-465, by means of particle bombardment. Agrobacteria-mediatedtransformations can be produced, for example, as described by Mlynarovaet al. (1994), Plant Cell Report 13: 282-285.

Example 7 Lipid Extraction from Seed

Plant material was first of all mechanically homogenized by means oftriturators in order to render it more amenable to extraction.

It was then heated to 100° C. for 10 min and after cooling on icesedimented again. The cell sediment was hydrolyzed with 1 M methanolicsulfuric acid and 2% dimethoxypropane for 1 h at 90° C. and the lipidswere transmethylated. The resultant fatty acid methyl esters (FAMEs)were finally extracted into petroleum ether. The extracted FAMEs wereanalyzed by gas-liquid chromatograph using a capillary column(Chrompack, WCOT fused silica. CP wax 52 CB. 25 m, 0.32 mm) and atemperature gradient of from 170° C. to 240° C. in 20 min and 5 min at240° C. The identity of the fatty acid methyl esters was confirmed bycomparison with corresponding FAME standards (Sigma). The identity andthe position of the double bond was further analyzed by means of GC-MSby suitable chemical derivatization of the FAME mixtures, e.g. to form4,4-dimethoxyoxazoline derivatives (Christie, 1998). The GC analyses ofthe fatty acid methyl esters obtained from the transgenic rape seedsexhibiting seed-specific expression of the Δ6-desaturase are presentedin Table 1. The transgenic rape seeds contained up to 5% of γ-linolenicacid in the seed.

Example 8 Expression of Δ6-Desaturases from Primulaceae in Yeast(Saccharomyces cerevisiae)

The open reading rasters of the Δ6-desaturases obtained fromMuscariodides vialii and Aleuritia farinosa were each cloned behind thegalactose-inducible GAL1 promoter of the yeast expression vector pYES2(Invitrogen). The open reading rasters were amplified by means of PCR.The interfaces used for cloning were KpnI and EcoRI.

Primer Design for M. vialii:

(SEQ ID NO: 14) ggtaccatggctaacaaatctcccacc (KpnI) (SEQ ID NO: 15)gaattcttagccgtgtgtgtggacggctt (EcoRI)

Primer Design for A. farinosa:

(SEQ ID NO: 14) ggtaccatggctaacaaatctcccacc (KpnI) (SEQ ID NO: 16)gaattctcacccgagagttttaagagct (EcoRI)

The vectors produced were used for expression in yeast. The substratespecificities were determined by feeding the transformed yeast strainswith α-linolenic acid and linoleic acid. The methodology used isdescribed, for example, in Napier and Michaelson, 2001, Lipids.36(8):761-766; Sayanova et al., 2001, Journal of Experimental Botany.52(360):1581-1585, Sperling et al., 2001, Arch. Biochem. Biophys.388(2):293-298 and Michaelson et al., 1998, FEBS Letters.439(3):215-218.

In order to compare the specificities the Δ6-desaturase from borage(Borago officinalis) was likewise expressed in yeast (Sayanova et al.,1999, Plant Physiol. 121(2):641-646).

TABLE 1 Expression in yeast. Comparison of substrate specificities of Δ6-de- saturases from borage, A. farinosa (SEQ ID NO: 1) and M. vialii(SEQ ID NO: 3). Construct Fatty Acid pYES2 Borage A. farinosa M. vialiiC_(16:0) 24.8 20 23.2 20.3 C_(16:1) ^(Δ9) 22.5 20 18.2 21.7 C_(18:0) 6.16.2 5.9 4.7 C_(18:1) ^(Δ9) 17.1 17.1 14.9 15.8 C_(18:2) ^(Δ, 9, 12) 13.811.5 12.8 15.3 C_(18:3) ^(Δ6, 9, 12) 0 5.5 5 1.4 C_(18:3) ^(Δ9, 12, 15)15.6 15.5 12.2 13.9 C_(18:4) ^(Δ6, 9, 12, 15) 0 3.9 7.7 6.8

TABLE 2 Conversion of linoleic acid by Δ 6-desaturase from borage andthe Δ 6-desaturases from Primulaceae (A. farinosa (SEQ ID NO: 1) and M.vialii (SEQ ID NO: 3)) by comparison with the conversion of α-linolenicacid. Construct pYES2 Borage A. farinosa M. vialii % by wt. of Δ 6-de-nd 11.10% 14.10% 8.80% saturated fatty acids in the total fatty acids %conversion of LA nd 32.30% 28.10% 8.40% to Δ 6 fatty acids % conversionof ALA nd 20.10% 38.70% 32.80%  to Δ 6 fatty acids Ratio n3:n6 0.71 1.54  4.8   LA = linoleic acid (=C_(18:2) ^(Δ9, 12))

Δ 6-desaturation produces C_(18:3) ^(Δ6, 9, 12) ALA = α-linolenic acid(=C_(18:3) ^(Δ9, 12, 15))

Δ 6-desaturation produces C_(18:4) ^(Δ) _(6, 9, 12, 15) nd = notdetermined

It may be gathered from the tables that the nucleic acid sequencesaccording to the invention encode Δ6-desaturases which are specific forω-3 fatty acids.

1. An isolated nucleic acid that encodes a polypeptide havingΔ6-desaturase activity, wherein the polypeptide exhibits higherΔ6-desaturase activity for ω-3 fatty acids as compared to ω-6 fattyacids, wherein said nucleic acid comprises a nucleotide sequenceselected from the group consisting of: a) the nucleotide sequence as setforth in SEQ ID NO: 3; b) a nucleotide sequence that encodes apolypeptide comprising the amino acid sequence as set forth in SEQ IDNO: 4; c) a nucleotide sequence having at least 75% homology to thenucleic acid sequence as set forth in SEQ ID NO: 3; and d) a nucleotidesequence that encodes a polypeptide comprising an amino acid sequencehaving at least 75% homology to the amino acid sequence as set forth inSEQ ID NO:
 4. 2. The isolated nucleic acid of claim 1, wherein thenucleic acid is derived from a plant.
 3. The isolated nucleic acid ofclaim 1, wherein the nucleic acid is derived from the generaMuscariodides or Aleuritia.
 4. The isolated nucleic acid of claim 1,wherein the nucleic acid comprises a nucleotide sequence selected fromthe group consisting of: a) a nucleotide sequence having at least 95%homology to the nucleic acid sequence as set forth in SEQ ID NO: 3; andb) a nucleotide sequence that encodes a polypeptide comprising an aminoacid sequence having at least 95% homology to the amino acid sequence asset forth in SEQ ID NO:
 4. 5. An amino acid sequence which is encoded bythe isolated nucleic acid of claim
 1. 6. A gene construct containing theisolated nucleic acid of claim 1, wherein said nucleic acid isfunctionally linked to one or more regulation signals.
 7. A vectorcontaining the isolated nucleic acid of claim
 1. 8. A transgenicnonhuman organism containing the isolated nucleic acid of claim
 1. 9.The transgenic nonhuman organism of claim 8, wherein the organism is amicroorganism, yeast, or plant.
 10. The transgenic nonhuman organism ofclaim 8, wherein the organism is a plant.
 11. A method for producingpolyunsaturated fatty acids, wherein the method comprises growing atransgenic organism which comprises the isolated nucleic acid of claim1, wherein, due to the activity of said Δ-6 desaturase, polyunsaturatedfatty acids are formed in said organism which exhibits an increasedcontent of ω-3 fatty acids compared to a non-transformed organism. 12.The method of claim 11, wherein in the stearidonic acid,eicosapentaenoic acid, or docosahexaenoic acid is produced.
 13. Themethod of claim 11, further comprising isolating polyunsaturated fattyacid molecules from said organism in the form of an oil, lipid, or afree fatty acid.
 14. The method of claim 11, wherein said organism is amicroorganism, a nonhuman animal, a yeast, or a plant.
 15. The method ofclaim 11, wherein said organism is a transgenic plant.
 16. The method ofclaim 13, wherein said polyunsaturated fatty acid molecules comprise aC₁₈ fatty acid having at least three double bonds.
 17. Oil, lipids, orfatty acids, or a fraction thereof, produced by the method of claim 11.18. A composition comprising the oil, lipids or fatty acids, or fractionthereof, of claim 17, further comprising polyunsaturated fatty acids.19. Animal feed, foodstuffs, cosmetics or pharmaceuticals comprising theisolated oil, lipids or fatty acids, or fraction thereof obtained by themethod of claim
 13. 20. The isolated nucleic acid of claim 1, whereinthe Δ6-desaturase activity toward ω-3 fatty acids as compared to ω-6fatty acids is higher by a factor of at least 1.5.
 21. The isolatednucleic acid of claim 20, wherein the factor is at least
 3. 22. Theisolated nucleic acid of claim 20, wherein the factor is at least
 5. 23.A vector containing the gene construct of claim
 6. 24. A transgenicnonhuman organism containing the gene construct of claim
 6. 25. The geneconstruct of claim 6, wherein the one or more regulation signalscomprise one or more promoter, terminator, or polyadenylation site. 26.The method of claim 11, which produces triglycerides having an increasedcontent of saturated or unsaturated fatty acids as compared to anuntransformed organism.
 27. The method of claim 11, wherein saidorganism has an increased content of polyunsaturated fatty acids ascompared to an untransformed organism without said nucleic acid.
 28. Themethod of claim 27, wherein said increased content of polyunsaturatedfatty acids is at least 10% increased as compared to an untransformedorganism without said nucleic acid.
 29. The method of claim 27, whereinsaid increased content of polyunsaturated fatty acids is at least 20%increased as compared to an untransformed organism without said nucleicacid.
 30. The method of claim 27, wherein said increased content ofpolyunsaturated fatty acids is at least 40% increased to anuntransformed organism without said nucleic acid.
 31. The method ofclaim 13, wherein said polyunsaturated fatty acid molecules comprisefatty acids having an increased content of Δ6-double bonds as comparedto an untransformed organism.
 32. A method for the production of animalfeed, foodstuffs, cosmetics, or pharmaceuticals, comprising culturingthe transgenic organism of claim 8; isolating polyunsaturated fatty acidmolecules from said organism in the form of an oil, lipid, or a freefatty acid; and utilizing the oil, lipid, or free fatty acid in theproduction of animal feed, foodstuffs, cosmetics, or pharmaceuticals.33. An amino acid sequence which is encoded by an isolated nucleic acidthat encodes a polypeptide having Δ6-desaturase activity, wherein thepolypeptide exhibits higher Δ6-desaturase activity for ω-3 fatty acidsas compared to ω-6 fatty acids, wherein said nucleic acid comprises anucleotide sequence selected from the group consisting of: a) thenucleotide sequence as set forth in SEQ ID NO: 1; b) a nucleotidesequence that encodes a polypeptide comprising the amino acid sequenceas set forth in SEQ ID NO: 2; and c) a nucleotide sequence that encodesa polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence as set forth in SEQ ID NO: 2,wherein the polypeptide has Δ6-desaturase activity, and the polypeptideexhibits higher Δ6-desaturase activity for ω-3 fatty acids as comparedto ω-6 fatty acids.